temperature and concentration dependent effects of combining DFP with DFO on FO formation from iron: citrate were next examined using HPLC, that allows certain identification of the FO complex when mixtures of the 2 chelators are utilized. When DFO was incubated with metal citrate at RT for approximately 24h, FO creation was again biphasic, taking over 24h to reach completion, consistent with the spectrophotometrically established kinetics of Figure 4. The fast phase was too fast to measure by this technique and had an amplitude of 3 uM FO. It can be seen that DFP enhanced the price of the slower second stage in a concentration order Avagacestat dependent manner, together with the maximum effect at 30uM DFP. However, even low concentrations of DFP boost the rate of FO development, consistent with the idea of DFP working as a shuttle at low concentrations. Whilst the rate of FO formation was maximally increased at 30uM DFP, another increase in DFP focus to 100 uM showed a little decrease in the rate of FO formation compared to that observed with 10 or 30 uM DFP, suggesting that DFP at higher concentrations may maintain the chelated iron and therefore slow its rate of shuttling to DFO. There was no significant difference Eumycetoma between any of the FO concentrations when comparing to DFO alone measured at zero time for any mixture of DFO and DFP. DFO plus all concentrations of DFP and major variations between DFO alone occurred in FO formation at all subsequent time points except where DFP was 3 uM. Here a significant big difference was seen after 2 h and at all subsequent time points. It may be seen that the price of the next phase of FO formation is temperature dependent both in the absence and presence of DFP. Ergo FO levels reach a 9. 4 uM after 8h at 37 C, while at RT this was 6. 4 uM after 8 h and only 9. 0 uM after 24h. In Lu AA21004 contrast to the slow phase, the amplitude of FO development in the fast phase was not dramatically influenced by the DFP levels tested. This period could not be accounted for by metal contamination in just about any of the reagents used, that has been determined as 0. 75 uM by injection of reaction mixtures where iron was omitted. As neither HPLC nor conventional spectrophotometry are appropriate to examine the fast phase of FO development, the rate of the approach was examined on the first 50 seconds of reaction using a stopped flow spectrometer. This covers the time range inaccessible within the traditional spectrophotometer and HPLC, representing the injection and mixing time for incubations carried out in these devices. The rate of this phase was more rapid for DFP iron complex formation than for DFO but the amplitude of iron chelation was similar at 50 seconds showing a similar amount of total available iron chelated by either chelator.